Phage display of peptides or proteins on filamentous phage particles is an in vitro technology which allows the selection of peptides or proteins with desired properties from large pools of variant peptides or proteins (McCafferty et al., Nature, 348: 552-554 (1990); Sidhu et al., Current Opinion in Biotechnology, 11: 610-616 (2000); Smith et al., Science, 228: 1315-1317 (1985)). Phage display may be used to display diverse libraries of peptides or proteins, including antibody fragments, such as Fabs in the antibody discovery field, on the surface of a filamentous phage particle which are then selected for binding to a particular antigen of interest. The antibody fragment may be displayed on the surface of the filamentous phage particle by fusing the gene for the antibody fragment to that of a phage coat protein, resulting in a phage particle that displays the encoded antibody fragment on its surface. This technology allows the isolation of antibody fragments with desired affinity to many antigens form a large phage library.
For phage-based antibody discovery, evaluation of selected antibody fragments and the properties of their cognate IgGs in functional assays (such as target binding, cell-based activity assays, in vivo half-life, etc.) requires reformatting of the Fab heavy chain (HC) and light chain (LC) sequences into a full-length IgG by subcloning the DNA sequences encoding the HC and LC out of the vector used for phage display and into mammalian expression vectors for IgG expression. The laborious process of subcloning dozens or hundreds of selected HC/LC pairs represents a major bottleneck in the phage-based antibody discovery process. Furthermore, since a substantial percentage of selected Fabs, once reformatted, fail to perform satisfactorily in initial screening assays, increasing the number of clones carried through this reformatting/screening process greatly increases the ultimate probability of success.
Here, we describe the generation of an expression and secretion system for driving expression of a Fab-phage fusion when transformed into E. coli, and of driving expression of a full-length IgG bearing the same Fab fragment when transfected into mammalian cells. We demonstrate that a mammalian signal sequence from the murine binding immunoglobulin protein (mBiP) (Haas et al., Immunoglobulin heavy chain binding protein, Nature, 306: 387-389 (1983); Munro et al., An Hsp70-like protein in the ER: identify with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein, Cell, 4:291-300 (1986) can drive efficient protein expression in both prokaryotic and eukaryotic cells. Using mammalian mRNA splicing to remove a synthetic intron containing a phage fusion peptide inserted within the hinge region of the human IgG1 HC, we are able to generate two distinct proteins in a host cell-dependent fashion: a Fab fragment fused to an adaptor peptide for phage display in E. coli and native human IgG1 in mammalian cells. This technology allows for the selection of Fab fragments that bind to an antigen of interest from a phage display library with subsequent expression and purification of the cognate full-length IgGs in mammalian cells without the need for subcloning.